Enhanced depletion of targeted cells with cd47 blockade and an immune costimulatory agonist

ABSTRACT

Methods are provided for targeting cells for depletion, including without limitation tumor cells, in a regimen comprising contacting the targeted cells with a combination of agents that modulate immunoregulatory signaling. Immunoregulatory modulating agents include (i) an agent that blockades CD47 activity; and (ii) an agent that agonizes an immune costimulatory molecule, e.g. CD137. The regimen may further comprise an agent that specifically binds to the target cell, e.g. an antibody or biologically active fragment or derivative thereof. The level of depletion of the targeted cell is enhanced relative to a regimen in which a single immunoregulatory modulating agent is used; and the effect may be synergistic relative to a regimen in which a single immunoregulatory modulating agent is used.

CROSS-REFERENCE

This application is a Continuation and claims the benefit of 371application Ser. No. 15/754,757, filed Feb. 23, 2018, which claims thebenefit of PCT Application No. PCT/US2016/049016, filed Aug. 26, 2016,which claims benefit of U.S. Provisional Patent Application Ser. No.62/210,279 filed Aug. 26, 2015, which applications are incorporatedherein by reference in their entirety.

Turnover of cells begins with the induction of an apoptotic program orother cellular changes that mark them for removal, and the subsequentrecognition of markers by phagocytes, including macrophages, dendriticcells, and the like. This process requires a specific and selectiveremoval of unwanted cells. Discrimination of the healthy from theunwanted/aged/dying cells display markers or ligands called “eat-me”signals, i.e. “altered self”, which can in turn be recognized byreceptors on the phagocytes. Healthy cells may display “don't eat-me”signals that actively inhibit phagocytosis; these signals are eitherdownregulated in the dying cells or present in an altered conformation.The cell surface protein CD47 on healthy cells and its engagement of aphagocyte receptor, SIRPα, constitutes a key “don't eat-me” signal thatcan turn off engulfment mediated by multiple modalities, includingapoptotic cell clearance and FcR mediated phagocytosis. Blocking theCD47 mediated engagement of SIRPα on a phagocyte, or the loss of CD47expression in knockout mice, can cause removal of live cells andnon-aged erythrocytes. Alternatively, blocking SIRPα recognition alsoallows engulfment of targets that are not normally phagocytosed.

CD47 is a broadly expressed transmembrane glycoprotein with a singleIg-like extracellular domain and five membrane spanning regions. CD47functions as a cellular ligand for SIRPα with binding mediated throughthe NH₂-terminal V-like domain of SIRPα. SIRPα is expressed primarily onmyeloid cells, including macrophages, granulocytes, myeloid dendriticcells (DCs), mast cells, and their precursors, including monocytes andhematopoietic stem cells. Structural determinants on SIRPα that mediateCD47 binding are discussed by Lee et al. (2007) J. Immunol.179:7741-7750; Hatherley et al. (2007) J. B. C. 282:14567-75; and therole of SIRPα cis dimerization in CD47 binding is discussed by Lee etal. (2010) J. B. C. 285:37953-63.

Immune cells such as T cells and NK cells are also regulated bysignaling pathways, including an antigen non-specific co-stimulatorysignal, which may be provided by molecules on antigen presenting cellsthat engage particular costimulatory receptors on the immune cells.Costimulation is crucial to the development of an effective immuneresponse of adaptive immunity.

One of the best characterized costimulatory receptors expressed by Tcells is CD28, which interacts with CD80 (B7-1) and CD86 (B7-2) on themembrane of APCs. CD28 is constitutively expressed on almost all Tcells, and is the major costimulatory receptor for naive T cells.Several members of the tumor necrosis factor receptor (TNFR) familyfunction after initial T cell activation to sustain T cell or NK cellresponses. The effects of these costimulatory TNFR family members canoften be functionally, temporally, or spatially segregated from those ofCD28 and from each other.

TNFR family members can recruit TNF receptor-associated factor (TRAF)adapter proteins and activate the nuclear factor KB (NF-κB) signalingpathway, making them fundamentally distinct from costimulators such asCD28 or ICOS. CD40 and its ligand, CD154, were the first costimulatorymolecules to be identified as members of the TNFR/TNF superfamily andare crucial for the functions of B cells and dendritic cells (DCs).Studies of the CD27/CD70, CD30/CD30L, OX40/OX40L, 4-1 BB (CD137)/4-1BBL, glucocorticoid-induced TNF receptor (GITR)/GITR ligand, herpesvirus entry mediator (HVEM)/(LIGHT) pathways indicate that theseTNFR/TNF family members provide important costimulatory signals. Withthe exception of CD27, the TNFR are expressed only upon T-cell or NKcell activation.

Recent work indicates that OX40/OX40L and CD137/CD137L interactions havekey roles in regulating the balance between effector and Treg responses.A major role of CD137 is for survival of activated and memory T cells,with preferential effects on CD8₊ T cells. However, when othercostimulatory signals are limiting, CD137 signals can cooperate withTCR-induced signals to enhance proliferation and development of effectorfunction. Upon Fc receptor triggering, human NK cells upregulate CD137,thereby enhancing the killing function of these activated NK cells.Agonistic anti-CD137 monoclonal antibodies (BMS-663513; Urelumab) arecurrently in clinical trials as a monotherapy, or combined with tumorspecific antibodies for the treatment of cancer.

SUMMARY OF THE INVENTION

Methods are provided for targeting cells for depletion, includingwithout limitation tumor cells, in a regimen comprising contacting thetargeted cells with a combination of agents that modulateimmunoregulatory signaling. Immunoregulatory modulating agents include(i) an agent that blockades CD47 activity; and (ii) an agent thatagonizes an immune costimulatory molecule, e.g. CD137. The regimen mayfurther comprise an agent that specifically binds to the target cell,e.g. an antibody or biologically active fragment or derivative thereof.The level of depletion of the targeted cell is enhanced relative to aregimen in which a single immunoregulatory modulating agent is used; andthe effect may be synergistic relative to a regimen in which a singleimmunoregulatory modulating agent is used.

The agents in the combination are administered concomitantly, i.e. eachagent is administered within about 7 days, 6 days, 5 days, 4 days, 3days, 2 days, 1 day or substantially simultaneously with respect to theother agent(s) in the combination. Administration may be repeated asnecessary for depletion of the targeted cell population.

In some embodiments the CD47 blockade is accomplished by administering asoluble SIRPα polypeptide, which may be a high affinity SIRPα variantpolypeptide. In other embodiments, antibodies specific for one or bothof SIRPα and CD47 are administered. In some embodiments thecostimulatory agonist is an antibody that selectively binds to thecostimulatory molecule, e.g. an agonist anti-CD137 antibody.

The contacting of a targeted cell may be performed in vivo, e.g. fortherapeutic purposes, and in vitro, e.g. for screening assays and thelike. In related embodiments, tumor cells, e.g. solid tumors such ascarcinomas, sarcomas, melanomas, etc.; leukemias; lymphomas, etc. aretargeted for depletion by contacting the immune cells, includingphagocytic cells, NK cells, T cells, etc. in proximity of the tumorcells with a combination of a CD47 blocking agent that is effective toblock the interaction between CD47 and SIRPα, and an agent that agonizesan immune costimulatory molecule, e.g. CD137. Optionally an agent thatspecifically binds to the targeted cell is included in the combination.In these aspects, the combination of immunoregulatory agents can becombined with monoclonal antibodies directed against one or moreadditional tumor cell markers, which compositions can be synergistic inenhancing phagocytosis and elimination of tumor cells as compared to theuse of single agents. The effective dose of the combined agentsincreases the depletion of the tumor cells.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following FIGURES.

FIG. 1. Mastocytoma survival curve.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods are provided for the targeted depletion of cells in a subject,where targeted cells are selectively ablated by a combination of agentsthat modulate immunoregulatory pathways in the subject. One agent thatmodifies immunoregulatory signaling blocks CD47 signaling. The secondagent is an agonist of an immune costimulatory molecule, e.g. CD137.

To facilitate an understanding of the invention, a number of terms aredefined below.

Before the present active agents and methods are described, it is to beunderstood that this invention is not limited to the particularmethodology, products, apparatus and factors described, as such methods,apparatus and formulations may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “adrug candidate” refers to one or mixtures of such candidates, andreference to “the method” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, formulations and methodologies whichare described in the publication and which might be used in connectionwith the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

Generally, conventional methods of protein synthesis, recombinant cellculture and protein isolation, and recombinant DNA techniques within theskill of the art are employed in the present invention. Such techniquesare explained fully in the literature, see, e.g., Maniatis, Fritsch &Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook,Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001);Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: PortableProtocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory;(1988).

Definitions

Anti-CD47 agent. CD47 is a broadly expressed transmembrane glycoproteinwith a single Ig-like domain and five membrane spanning regions, whichfunctions as a cellular ligand for SIRPα with binding mediated throughthe NH₂-terminal V-like domain of SIRPα. SIRPα is expressed primarily onmyeloid cells, including macrophages, granulocytes, myeloid dendriticcells (DCs), mast cells, and their precursors, including hematopoieticstem cells. Structural determinants on SIRPα that mediate CD47 bindingare discussed by Lee et al. (2007) J. Immunol. 179:7741-7750; Hatherleyet al. (2008) Mol Cell. 31(2):266-77; Hatherley et al. (2007) J. B. C.282:14567-75; and the role of SIRPα cis dimerization in CD47 binding isdiscussed by Lee et al. (2010) J. B. C. 285:37953-63. In keeping withthe role of CD47 to inhibit phagocytosis of normal cells, there isevidence that it is transiently upregulated on hematopoietic stem cells(HSCs) and progenitors just prior to and during their migratory phase,and that the level of CD47 on these cells determines the probabilitythat they are engulfed in vivo.

As used herein, the term “anti-CD47 agent” or “agent that provides forCD47 blockade” refers to any agent that reduces the binding of CD47(e.g., on a target cell) to SIRPα (e.g., on a phagocytic cell).Non-limiting examples of suitable anti-CD47 reagents include SIRPαreagents, including without limitation high affinity SIRPα polypeptides,anti-SIRPα antibodies, soluble CD47 polypeptides, and anti-CD47antibodies or antibody fragments. In some embodiments, a suitableanti-CD47 agent (e.g. an anti-CD47 antibody, a SIRPα reagent, etc.)specifically binds CD47 to reduce the binding of CD47 to SIRPα.

In some embodiments, a suitable anti-CD47 agent (e.g., an anti-SIRPαantibody, a soluble CD47 polypeptide, etc.) specifically binds SIRPα toreduce the binding of CD47 to SIRPα. A suitable anti-CD47 agent thatbinds SIRPα does not activate SIRPα (e.g., in the SIRPα-expressingphagocytic cell). The efficacy of a suitable anti-CD47 agent can beassessed by assaying the agent. In an exemplary assay, target cells areincubated in the presence or absence of the candidate agent. An agentfor use in the methods of the invention will up-regulate phagocytosis byat least 5% (e.g., at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%, at least 120%, at least 140%, at least 160%, atleast 180%, at least 200%, at least 500%, at least 1000%) compared tophagocytosis in the absence of the agent. Similarly, an in vitro assayfor levels of tyrosine phosphorylation of SIRPα will show a decrease inphosphorylation by at least 5% (e.g., at least 10%, at least 15%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or 100%) compared tophosphorylation observed in absence of the candidate agent.

In some embodiments, the anti-CD47 agent does not activate CD47 uponbinding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) may occur (Manna and Frazier, Cancer Research,64, 1026-1036, Feb. 1 2004). Thus, in some embodiments, the anti-CD47agent does not directly induce cell death of a CD47-expressing cell.

SIRPα reagent. A SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, which normally liesbetween the signal sequence and the transmembrane domain, or a fragmentthereof that retains the binding activity. A suitable SIRPα reagentreduces (e.g., blocks, prevents, etc.) the interaction between thenative proteins SIRPα and CD47. The SIRPα reagent will usually compriseat least the dl domain of SIRPα.

In some embodiments, a subject anti-CD47 agent is a “high affinity SIRPαreagent”, which includes SIRPα-derived polypeptides and analogs thereof(e.g., CV1-hIgG4, and CV1 monomer). High affinity SIRPα reagents aredescribed in international application PCT/US13/21937, which is herebyspecifically incorporated by reference. High affinity SIRPα reagents arevariants of the native SIRPα protein. The amino acid changes thatprovide for increased affinity are localized in the dl domain, and thushigh affinity SIRPα reagents comprise a dl domain of human SIRPα, withat least one amino acid change relative to the wild-type sequence withinthe dl domain. Such a high affinity SIRPα reagent optionally comprisesadditional amino acid sequences, for example antibody Fc sequences;portions of the wild-type human SIRPα protein other than the dl domain,including without limitation residues 150 to 374 of the native proteinor fragments thereof, usually fragments contiguous with the dl domain;and the like. High affinity SIRPα reagents may be monomeric ormultimeric, i.e. dimer, trimer, tetramer, etc. In some embodiments, ahigh affinity SIRPα reagent is soluble, where the polypeptide lacks theSIRPα transmembrane domain and comprises at least one amino acid changerelative to the wild-type SIRPα sequence, and wherein the amino acidchange increases the affinity of the SIRPα polypeptide binding to CD47,for example by decreasing the off-rate by at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 500-fold, ormore.

Optionally the SIRPα reagent is a fusion protein, e.g., fused in framewith a second polypeptide. In some embodiments, the second polypeptideis capable of increasing the size of the fusion protein, e.g., so thatthe fusion protein will not be cleared from the circulation rapidly. Insome embodiments, the second polypeptide is part or whole of animmunoglobulin Fc region. The Fc region aids in phagocytosis byproviding an “eat me” signal, which enhances the block of the “don't eatme” signal provided by the high affinity SIRPα reagent. In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding. Some anti-CD47 antibodies do not reduce the binding of CD47 toSIRPα (and are therefore not considered to be an “anti-CD47 agent”herein) and such an antibody can be referred to as a “non-blockinganti-CD47 antibody.” A suitable anti-CD47 antibody that is an “anti-CD47agent” can be referred to as a “CD47-blocking antibody”. Non-limitingexamples of suitable antibodies include clones B6H12, 5F9, 8B6, and C3(for example as described in International Patent Publication WO2011/143624, herein specifically incorporated by reference). Suitableanti-CD47 antibodies include fully human, humanized or chimeric versionsof such antibodies. Humanized antibodies, for example comprising an IgG4Fc region, (e.g., hu5F9-G4) are especially useful for in vivoapplications in humans due to their low antigenicity. Similarlycaninized, felinized, etc. antibodies are especially useful forapplications in dogs, cats, and other species respectively. Antibodiesof interest include humanized antibodies, or caninized, felinized,equinized, bovinized, porcinized, etc., antibodies, and variantsthereof.

Anti-SIRPα antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds SIRPα (i.e., an anti-SIRPα antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). Suitableanti-SIRPα antibodies can bind SIRPα without activating or stimulatingsignaling through SIRPα because activation of SIRPα would inhibitphagocytosis. Instead, suitable anti-SIRPα antibodies facilitate thepreferential phagocytosis of inflicted cells over normal cells. Thosecells that express higher levels of CD47 (e.g., infected cells) relativeto other cells (non-infected cells) will be preferentially phagocytosed.Thus, a suitable anti-SIRPα antibody specifically binds SIRPα (withoutactivating/stimulating enough of a signaling response to inhibitphagocytosis) and blocks an interaction between SIRPα and CD47. Suitableanti-SIRPα antibodies include fully human, humanized or chimericversions of such antibodies. Humanized antibodies are especially usefulfor in vivo applications in humans due to their low antigenicity.Similarly caninized, felinized, etc. antibodies are especially usefulfor applications in dogs, cats, and other species respectively.Antibodies of interest include humanized antibodies, or caninized,felinized, equinized, bovinized, porcinized, etc., antibodies, andvariants thereof.

Soluble CD47 polypeptides. In some embodiments, a subject anti-CD47agent is a soluble CD47 polypeptide that specifically binds SIRPα andreduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). A suitablesoluble CD47 polypeptide can bind SIRPα without activating orstimulating signaling through SIRPα because activation of SIRPα wouldinhibit phagocytosis. Instead, suitable soluble CD47 polypeptidesfacilitate the preferential phagocytosis of infected cells overnon-infected cells. Those cells that express higher levels of CD47(e.g., infected cells) relative to normal, non-target cells (normalcells) will be preferentially phagocytosed. Thus, a suitable solubleCD47 polypeptide specifically binds SIRPα without activating/stimulatingenough of a signaling response to inhibit phagocytosis.

In some cases, a suitable soluble CD47 polypeptide can be a fusionprotein (for example as structurally described in US Patent PublicationUS20100239579, herein specifically incorporated by reference). However,only fusion proteins that do not activate/stimulate SIRPα are suitablefor the methods provided herein. Suitable soluble CD47 polypeptides alsoinclude any peptide or peptide fragment comprising variant or naturallyexisting CD47 sequences (e.g., extracellular domain sequences orextracellular domain variants) that can specifically bind SIRPα andinhibit the interaction between CD47 and SIRPα without stimulatingenough SIRPα activity to inhibit phagocytosis.

In certain embodiments, soluble CD47 polypeptide comprises theextracellular domain of CD47, including the signal peptide, such thatthe extracellular portion of CD47 is typically 142 amino acids inlength. The soluble CD47 polypeptides described herein also include CD47extracellular domain variants that comprise an amino acid sequence atleast 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% (or any percentidentity not specifically enumerated between 65% to 100%), whichvariants retain the capability to bind to SIRPα without stimulatingSIRPα signaling.

In certain embodiments, the signal peptide amino acid sequence may besubstituted with a signal peptide amino acid sequence that is derivedfrom another polypeptide (e.g., for example, an immunoglobulin orCTLA4). For example, unlike full-length CD47, which is a cell surfacepolypeptide that traverses the outer cell membrane, the soluble CD47polypeptides are secreted; accordingly, a polynucleotide encoding asoluble CD47 polypeptide may include a nucleotide sequence encoding asignal peptide that is associated with a polypeptide that is normallysecreted from a cell.

In other embodiments, the soluble CD47 polypeptide comprises anextracellular domain of CD47 that lacks the signal peptide. As describedherein, signal peptides are not exposed on the cell surface of asecreted or transmembrane protein because either the signal peptide iscleaved during translocation of the protein or the signal peptideremains anchored in the outer cell membrane (such a peptide is alsocalled a signal anchor). The signal peptide sequence of CD47 is believedto be cleaved from the precursor CD47 polypeptide in vivo.

In other embodiments, a soluble CD47 polypeptide comprises a CD47extracellular domain variant. Such a soluble CD47 polypeptide retainsthe capability to bind to SIRPα without stimulating SIRPα signaling. TheCD47 extracellular domain variant may have an amino acid sequence thatis at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical(which includes any percent identity between any one of the describedranges) to the native CD47 sequence.

Immunoregulatory signaling molecules. In addition to the CD47/SIRPαaxis, immunoregulatory signaling molecules may include costimulatorypolypeptides expressed on immune cells, e.g. T cells, NK cells, antigenpresenting cells, etc. Activation, i.e. agonism, of the costimulatorymolecule enhances the effector cell function. Many such costimulatorymolecules are members of the tumor necrosis factor receptor family(TNFR), e.g. OX40, GITR, CD30, ICOS, etc. TNFR-related molecules do nothave any known enzymatic activity and depend on the recruitment ofcytoplasmic proteins for the activation of downstream signalingpathways.

A costimulatory molecule of interest is CD137, which may also bereferred to as Ly63, ILA or 4-1 BB, and which is a member of the tumornecrosis factor (TNF) receptor family. Members of this receptor familyand their structurally related ligands are important regulators of awide variety of physiologic processes and play an important role in theregulation of immune responses. CD137 is expressed by activated NKcells, T and B lymphocytes and monocytes/macrophages. The gene encodes a255-amino acid protein with 3 cysteine-rich motifs in the extracellulardomain (characteristic of this receptor family), a transmembrane region,and a short N-terminal cytoplasmic portion containing potentialphosphorylation sites. Expression in primary cells is strictlyactivation dependent. The ligand for the receptor is TNFSF9. Human CD137is reported to bind only to its ligand. Agonists include the nativeligand (TNFSF9), aptamers (see McNamara et al. (2008) J. Clin. Invest.118: 376-386), and antibodies.

Two fully humanized mAbs of CD137, urelumab (BMS-663513) andPF-05082566, have been developed for clinical use. Urelumab is a fullyhuman IgG4 mAb, and PF-05082566 is a fully human IgG2 mAb.

CD134. OX40 (CD134) and its binding partner, OX40L (CD252), are membersof the TNFR super-family. OX40 expression is induced following TCR/CD3cross-linking, and by the presence of inflammatory cytokines, includingIL-1, IL-2, and TNF-α. In humans, a substantial proportion oftumor-infiltrating CD4 T cells express OX40. Similarly, activatedperipheral CD8 T cells have also been shown to express OX40. Ligation ofOX40 on CD8 and conventional (non-regulatory) CD4 T cells, using eitherits natural ligand (OX40L) or agonist antibodies, promotes theirsurvival and expansion.

Treatment with agonist anti-OX40 monoclonal antibodies (mAbs) along withTCR stimulation in wild-type animals induces expansion, differentiation,and increased survival of CD4 and CD8 T cells. Anti-OX40 administrationcan overcome CD8 T cell tolerance to a self-antigen and restored theircytotoxic activity, highlighting the therapeutic potential for OX40agonists. This is of particular importance for patients with cancer, asT cell tolerance to the tumor is a major obstacle for therapeuticmodalities.

The use of anti-OX40 monotherapy was tested in a Phase 1 trial inpatients with solid tumors. Phase 1 clinical trials investigating OX40agonists including NCT02318394, NCT02205333, and NCT02221960, with thebiological MED16383.

Agonistic OX40 agents can enhance the efficacy of anti-CD47 agents.Agonistic OX40 agents may be administered substantially simultaneouslywith anti-CD47 agents; or may be administered prior to and concurrentlywith treatment with anti-CD47 to simulate priming of tumor-specific Tcell clones that can be expanded through the OX40 agent.

CD30. The transmembrane receptor CD30 (TNFRSF8) and its ligand CD30L(CD153, TNFSF8) are members of the tumor necrosis factor (TNF)superfamily and display restricted expression in subpopulations ofactivated immune cells. CD30 is a type I transmembrane glycoprotein ofthe TNF receptor superfamily. The ligand for CD30 is CD30L (CD153). Thebinding of CD30 to CD30L mediates pleiotropic effects including cellproliferation, activation, differentiation, and apoptotic cell death.Antibodies in clinical trials for cancer include CD30 agonists SGN-30;XmAb2513 and MDX-1401.

GITR. Glucocorticoid-Induced TNFR-Related (GITR) protein belongs totumor necrosis factor receptor/tumor necrosis factor superfamily andstimulates both the acquired and innate immunity. It is expressed inseveral cells and tissues, including T and Natural Killer (NK) cells andis activated by its ligand, GITRL, mainly expressed on antigenpresenting cells and endothelial cells. GITR/GITRL system participatesin the development of autoimmune/inflammatory responses and potentiatesresponse to infection and tumors by mechanisms including NK-cellco-activation. Antibodies in clinical trials include the GITR agonistTRX518.

Inducible costimulator (ICOS). ICOS is a member of the CD28 family. ICOSexpression, may be readily detectable resting, but it upregulated uponactivation. ICOS and ICOS-L appear to be a monogamous pair. ICOScostimulation enhances effector functions. ICOS specific agonistantibodies GSK3359609 or JTX-2011 target and bind to ICOS expressed ontumor infiltrating CD4-positive T cells. This stimulates ICOS-positiveT-cell proliferation, enhances cytotoxic T-lymphocyte (CTL) survival andincreases CTL-mediated immune responses against tumor cells. ICOS, aT-cell specific, CD28-superfamily costimulatory molecule and immunecheckpoint protein, is normally expressed on certain activated T cellsand plays a key role in the proliferation and activation of T cells.

Agonists includes the native ligands, as described above, aptamers,antibodies specific for an inducible costimulatory molecule thatactivate the receptor, and derivatives, variants, and biologicallyactive fragments of antibodies that selectively bind to a costimulatorymolecule. A “variant” polypeptide means a biologically activepolypeptide as defined below having less than 100% sequence identitywith a native sequence polypeptide. Such variants include polypeptideswherein one or more amino acid residues are added at the N- orC-terminus of, or within, the native sequence; from about one to fortyamino acid residues are deleted, and optionally substituted by one ormore amino acid residues; and derivatives of the above polypeptides,wherein an amino acid residue has been covalently modified so that theresulting product has a non-naturally occurring amino acid. Ordinarily,a biologically active variant will have an amino acid sequence having atleast about 90% amino acid sequence identity with a native sequencepolypeptide, preferably at least about 95%, more preferably at leastabout 99%. The variant polypeptides can be naturally or non-naturallyglycosylated, i.e., the polypeptide has a glycosylation pattern thatdiffers from the glycosylation pattern found in the correspondingnaturally occurring protein.

Fragments of the ligand or antibodies specific for a costimulatorymolecule, particularly biologically active fragments and/or fragmentscorresponding to functional domains, are of interest. Fragments ofinterest will typically be at least about 10 aa to at least about 15 aain length, usually at least about 50 aa in length, but will usually notexceed about 200 aa in length, where the fragment will have a contiguousstretch of amino acids that is identical to the polypeptide from whichit is derived. A fragment “at least 20 aa in length,” for example, isintended to include 20 or more contiguous amino acids from, for example,an antibody specific for CD137, or from TNFSF9. In this context “about”includes the particularly recited value or a value larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids. The protein variants describedherein are encoded by polynucleotides that are within the scope of theinvention. The genetic code can be used to select the appropriate codonsto construct the corresponding variants. The polynucleotides may be usedto produce polypeptides, and these polypeptides may be used to produceantibodies by known methods. A “fusion” polypeptide is a polypeptidecomprising a polypeptide or portion (e.g., one or more domains) thereoffused or bonded to heterologous polypeptide.

In some embodiments, the costimulatory molecule agonist is an antibody.The term “antibody” or “antibody moiety” is intended to include anypolypeptide chain-containing molecular structure with a specific shapethat fits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. Antibodies utilized in the present inventionmay be polyclonal antibodies, although monoclonal antibodies arepreferred because they may be reproduced by cell culture orrecombinantly, and can be modified to reduce their antigenicity.

In some embodiments, administration of a combination of agents of theinvention is combined with an effective dose of an agent that increasespatient hematocrit, for example erythropoietin stimulating agents (ESA).Such agents are known and used in the art, including, for example,Aranesp® (darbepoetin alfa), Epogen® NF/Procrit® NF (epoetin alfa),Omontys® (peginesatide), Procrit®, etc.

Other combination therapies include administration with cell-specificantibodies, for example antibodies selective for tumor cell markers,radiation, surgery, and/or hormone deprivation (Kwon et al., Proc. Natl.Acad. Sci U.S.A., 96: 15074-9, 1999). A number of antibodies arecurrently in clinical use for the treatment of cancer, and others are invarying stages of clinical development. Antibodies of interest for themethods of the invention may act through ADCC, and are typicallyselective for tumor cells, although one of skill in the art willrecognize that some clinically useful antibodies do act on non-tumorcells, e.g. CD20.

A number of antibodies are currently in clinical use for the treatmentof cancer, and others are in varying stages of clinical development. Forexample, there are a number of antigens and corresponding monoclonalantibodies for the treatment of B cell malignancies. One target antigenis CD20. Rituximab is a chimeric unconjugated monoclonal antibodydirected at the CD20 antigen. CD20 has an important functional role in Bcell activation, proliferation, and differentiation. The CD52 antigen istargeted by the monoclonal antibody alemtuzumab, which is indicated fortreatment of chronic lymphocytic leukemia. CD22 is targeted by a numberof antibodies, and has recently demonstrated efficacy combined withtoxin in chemotherapy-resistant hairy cell leukemia. Two new monoclonalantibodies targeting CD20, tositumomab and ibritumomab, have beensubmitted to the Food and Drug Administration (FDA). These antibodiesare conjugated with radioisotopes. Alemtuzumab (Campath) is used in thetreatment of chronic lymphocytic leukemia; Gemtuzumab (Mylotarg) findsuse in the treatment of acute myelogenous leukemia; Ibritumomab(Zevalin) finds use in the treatment of non-Hodgkin's lymphoma;Panitumumab (Vectibix) finds use in the treatment of colon cancer.

The CD52 antigen is targeted by the monoclonal antibody alemtuzumab,which is indicated for treatment of chronic lymphocytic leukemia; coloncancer and lung cancer. CD22 is targeted by a number of antibodies, andhas recently demonstrated efficacy combined with toxin inchemotherapy-resistant hairy cell leukemia.

Gemtuzumab (Mylotarg) finds use in the treatment of acute myelogenousleukemia; Ibritumomab (Zevalin) finds use in the treatment ofnon-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use in thetreatment of colon cancer.

Cetuximab (Erbitux) is also of interest for use in the methods of theinvention. The antibody binds to the EGF receptor (EGFR), and has beenused in the treatment of solid tumors including colon cancer andsquamous cell carcinoma of the head and neck (SCCHN).

Monoclonal antibodies useful in the methods of the invention that havebeen used in solid tumors include, without limitation, edrecolomab andtrastuzumab (herceptin). Edrecolomab targets the 17-1A antigen seen incolon and rectal cancer, and has been approved for use in Europe forthese indications. Trastuzumab targets the HER-2/neu antigen. Thisantigen is seen on 25% to 35% of breast cancers. Cetuximab (Erbitux) isalso of interest for use in the methods of the invention. The antibodybinds to the EGF receptor (EGFR), and has been used in the treatment ofsolid tumors including colon cancer and squamous cell carcinoma of thehead and neck (SCCHN).

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies) and heteroconjugate antibodies. The term“antibody” also includes antigen binding forms of antibodies, includingfragments with antigen-binding capability (e.g., Fab′, F(ab′)₂, Fab, Fvand rIgG. The term also refers to recombinant single chain Fv fragments(scFv). The term antibody also includes bivalent or bispecificmolecules, diabodies, triabodies, and tetrabodies.

Selection of antibodies may be based on a variety of criteria, includingselectivity, affinity, cytotoxicity, etc. The phrase “specifically (orselectively) binds” to an antibody or “specifically (or selectively)immunoreactive with,” when referring to a protein or peptide, refers toa binding reaction that is determinative of the presence of the protein,in a heterogeneous population of proteins and other biologics. Thus,under designated immunoassay conditions, the specified antibodies bindto a particular protein sequences at least two times the background andmore typically more than 10 to 100 times background. In general,antibodies of the present invention bind antigens on the surface oftarget cells in the presence of effector cells (such as natural killercells or macrophages). Fc receptors on effector cells recognize boundantibodies. The cross-linking of Fc receptors signals the effector cellsto kill the target cells by cytolysis or apoptosis. In one embodiment,the induction is achieved via antibody-dependent cellular cytotoxicity(ADCC).

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, or by immunizing ananimal with the antigen or with DNA encoding the antigen. Methods ofpreparing polyclonal antibodies are known to the skilled artisan. Theantibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods. In a hybridomamethod, an appropriate host animal is typically immunized with animmunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes may be immunized in vitro. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell.

Human antibodies can be produced using various techniques known in theart, including phage display libraries. Similarly, human antibodies canbe made by introducing of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire.

Antibodies also exist as a number of well-characterized fragmentsproduced by digestion with various peptidases. Thus pepsin digests anantibody below the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H1) by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region, therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially Fab with part of the hinge region. While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such fragments may be synthesized denovo either chemically or by using recombinant DNA methodology. Thus,the term antibody, as used herein, also includes antibody fragmentseither produced by the modification of whole antibodies, or thosesynthesized de novo using recombinant DNA methodologies (e.g., singlechain Fv) or those identified using phage display libraries.

A “humanized antibody” is an immunoglobulin molecule which containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Antibodies of interest may be tested for their ability to induce ADCC(antibody-dependent cellular cytotoxicity). Antibody-associated ADCCactivity can be monitored and quantified through detection of either therelease of label or lactate dehydrogenase from the lysed cells, ordetection of reduced target cell viability (e.g. annexin assay). Assaysfor apoptosis may be performed by terminal deoxynucleotidyltransferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay(Lazebnik et al., Nature: 371, 346 (1994). Cytotoxicity may also bedetected directly by detection kits known in the art, such asCytotoxicity Detection Kit from Roche Applied Science (Indianapolis,Ind.).

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, including pet andlaboratory animals, e.g. mice, rats, rabbits, etc. Thus the methods areapplicable to both human therapy and veterinary applications. In oneembodiment the patient is a mammal, preferably a primate. In otherembodiments the patient is human.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” encompass, without limitation,individuals having cancer. Subjects may be human, but also include othermammals, particularly those mammals useful as laboratory models forhuman disease, e.g. mouse, rat, etc.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. Cells of interestfor detection, analysis, or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. Cancers of virtually every tissue are known. Thephrase “cancer burden” refers to the quantum of cancer cells or cancervolume in a subject. Reducing cancer burden accordingly refers toreducing the number of cancer cells or the cancer volume in a subject.The term “cancer cell” as used herein refers to any cell that is acancer cell or is derived from a cancer cell e.g. clone of a cancercell. Many types of cancers are known to those of skill in the art,including solid tumors such as carcinomas, sarcomas, glioblastomas,melanomas, lymphomas, myelomas, etc., and circulating cancers such asleukemias. Examples of cancer include but are not limited to, ovariancancer, breast cancer, colon cancer, lung cancer, prostate cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, headand neck cancer, and brain cancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas cancer cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc. The term “biological sample” encompasses a clinicalsample, and also includes tissue obtained by surgical resection, tissueobtained by biopsy, cells in culture, cell supernatants, cell lysates,tissue samples, organs, bone marrow, blood, plasma, serum, and the like.A “biological sample” includes a sample obtained from a patient's cancercell, e.g., a sample comprising polynucleotides and/or polypeptides thatis obtained from a patient's cancer cell (e.g., a cell lysate or othercell extract comprising polynucleotides and/or polypeptides); and asample comprising cancer cells from a patient. A biological samplecomprising a cancer cell from a patient can also include non-cancerouscells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of breast cancer, prostate cancer,or other type of cancer.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of cancer-attributable death or progression, includingrecurrence, metastatic spread, and drug resistance, of a neoplasticdisease, such as ovarian cancer. The term “prediction” is used herein torefer to the act of foretelling or estimating, based on observation,experience, or scientific reasoning. In one example, a physician maypredict the likelihood that a patient will survive, following surgicalremoval of a primary tumor and/or chemotherapy for a certain period oftime without cancer recurrence.

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure, for the purposesof obtaining an effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of effecting a partial or complete cure fora disease and/or symptoms of the disease. “Treatment,” as used herein,may include treatment of a tumor in a mammal, particularly in a human,and includes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of an cancer, including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the disease condition more tolerable to the patient;slowing in the rate of degeneration or decline; or making the finalpoint of degeneration less debilitating. The treatment or ameliorationof symptoms can be based on objective or subjective parameters;including the results of an examination by a physician. Accordingly, theterm “treating” includes the administration of the compounds or agentsof the present invention to prevent or delay, to alleviate, or to arrestor inhibit development of the symptoms or conditions associated withcancer or other diseases. The term “therapeutic effect” refers to thereduction, elimination, or prevention of the disease, symptoms of thedisease, or side effects of the disease in the subject.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

In addition to cancer therapies, the combination of agents of theinvention are useful in other therapies in which monoclonal antibodiesare administered for the purpose of depleting cells, e.g. in thetreatment of inflammatory diseases by depletion of immune cells. Forsuch purposes the combination of agents of the invention is administeredin combination with a therapeutic antibody, e.g. with rituximab fordepletion of B cells in inflammatory diseases and autoimmune conditions;alemtuzumab for multiple sclerosis; OKT3 for immunosuppression; othersfor bone marrow transplant conditioning; and the like.

“Concomitant administration” of a cancer therapeutic drug, ESA ortumor-directed antibody with a pharmaceutical composition of the presentinvention means administration with the high affinity CD47 reagent atsuch time that both the drug, ESA or antibody and the composition of thepresent invention will have a therapeutic effect. Such concomitantadministration may involve concurrent (i.e. at the same time), prior, orsubsequent administration of the drug, ESA or antibody with respect tothe administration of a compound of the invention. A person of ordinaryskill in the art would have no difficulty determining the appropriatetiming, sequence and dosages of administration for particular drugs andcompositions of the present invention.

As used herein, the phrase “disease-free survival,” refers to the lackof such tumor recurrence and/or spread and the fate of a patient afterdiagnosis, with respect to the effects of the cancer on the life-span ofthe patient. The phrase “overall survival” refers to the fate of thepatient after diagnosis, despite the possibility that the cause of deathin a patient is not directly due to the effects of the cancer. Thephrases, “likelihood of disease-free survival”, “risk of recurrence” andvariants thereof, refer to the probability of tumor recurrence or spreadin a patient subsequent to diagnosis of cancer, wherein the probabilityis determined according to the process of the invention.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

Methods of Use

Methods are provided for treating, reducing or preventing cancer,including without limitation lymphomas, leukemias, carcinomas,melanomas, glioblastomas, sarcomas, myelomas, etc. as primary ormetastatic cancers, by n a regimen comprising contacting the targetedcells with a combination of agents that modulate immunoregulatorysignaling. Immunoregulatory modulating agents include (i) an agent thatblockades CD47 activity; and (ii) an agent that agonizes an immunecostimulatory molecule, e.g. CD137, thereby increasing in vivophagocytosis of the tumor cells. The regimen may further comprise anagent that specifically binds to the target cell, e.g. an antibody orbiologically active fragment or derivative thereof. Such methods includeadministering to a subject in need of treatment a therapeuticallyeffective amount or an effective dose of the combined agents of theinvention, including without limitation combinations of the reagent witha chemotherapeutic drug, a tumor-specific antibody, or an ESA.

Effective doses of the combined agents of the present invention, e.g.for the treatment of cancer, vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but nonhuman mammals mayalso be treated, e.g. companion animals such as dogs, cats, horses,etc., laboratory mammals such as rabbits, mice, rats, etc., and thelike. Treatment dosages can be titrated to optimize safety and efficacy.

In some embodiments, the therapeutic dosage of each agent may range fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once every two weeks or once a month oronce every 3 to 6 months. Therapeutic entities of the present inventionare usually administered on multiple occasions. Intervals between singledosages can be weekly, monthly or yearly. Intervals can also beirregular as indicated by measuring blood levels of the therapeuticentity in the patient. Alternatively, therapeutic entities of thepresent invention can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the polypeptidein the patient.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

In still other embodiments, methods of the present invention includetreating, reducing or preventing tumor growth, tumor metastasis or tumorinvasion of cancers including lymphomas, leukemias, carcinomas,melanomas, glioblastomas, sarcomas, myelomas, etc. For prophylacticapplications, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk ofdisease in an amount sufficient to eliminate or reduce the risk, lessenthe severity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease.

Compositions for the treatment of cancer can be administered byparenteral, topical, intravenous, intratumoral, oral, subcutaneous,intraarterial, intracranial, intraperitoneal, intranasal orintramuscular means. A typical route of administration is intravenous orintratumoral, although other routes can be equally effective.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the combined agents described herein can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. The data obtained from these cell culture assays and animalstudies can be used in formulating a dosage range that is not toxic foruse in human. The dosage of the proteins described herein liespreferably within a range of circulating concentrations that include theeffective dose with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition.

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include, but are notlimited to, powder, tablets, pills, capsules and lozenges. It isrecognized that compositions of the invention when administered orally,should be protected from digestion. This is typically accomplishedeither by complexing the molecules with a composition to render themresistant to acidic and enzymatic hydrolysis, or by packaging themolecules in an appropriately resistant carrier, such as a liposome or aprotection barrier. Means of protecting agents from digestion are wellknown in the art.

The compositions for administration will commonly comprise an antibodyor other ablative agent dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions maybe sterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of active agentin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs (e.g., Remington's Pharmaceutical Science (15th ed.,1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics(Hardman et al., eds., 1996)).

Also within the scope of the invention are kits comprising thecompositions (e.g., agonist of costimulatory molecules; anti-CD47agents, optionally antibodies that specifically bind to he targetedcells, and formulations thereof) of the invention and instructions foruse. The kit can further contain a least one additional reagent, e.g. achemotherapeutic drug, anti-tumor antibody, ESA, etc. Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

The compositions can be administered for therapeutic treatment.Compositions are administered to a patient in an amount sufficient tosubstantially ablate targeted cells, as described above. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.” Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. The particular dose required for a treatmentwill depend upon the medical condition and history of the mammal, aswell as other factors such as age, weight, gender, administration route,efficiency, etc.

EXPERIMENTAL Example 1 Combination of CD47 Blockade and CD137 Agonism inTargeting Cells for Immunodepletion

As described in the co-pending international application claimingpriority to U.S. provisional application 62/041,989, herein specificallyincorporated by reference, depletion of hematopoietic cells expressingCD117 is significantly enhanced by administering a combination of ananti-CD47 agent, an agonist of CD137, and an antibody targeted to CD117;relative to a comparable treatment in which an anti-CD47 agent isadministered with anti-CD117 in the absence of a CD137 agonist. Thesedata demonstrate the principle of combining activities to deplete atargeted cell population, which principle is herein extended totargeting depletion of tumor cells.

Shown in FIG. 1, 8 week old DBA/2J mice were injected with p815 mousemastocytoma cell line. 3 mice were injected with 250,000 untreated p815cells incubated for two hours at 37 degree C., and administered viaretro-orbital injections. 2 mice were injected with 250,000 p815 cellsincubated with 100 ug/ml anti-c-Kit (ACK2) antibody+100 ug/ml Clone 3(MIAP 410, anti-CD47 antibody) for two hours at 37 degree C., theninjected RO. Mice were given IP injection of 500 ug anti-CD137 (Lob12.3)day 0, 2 and 4.

Mice were assessed for death, as P815 cells are not GFP+. One controluntreated mouse died 4 weeks after the RO injection, another mouse died12 weeks post injection. Both combination therapy treated test groupmice were alive over 12 weeks post injection.

Example 2 In Vitro Synergy Experiment

An ADCC assay is performed using mouse or human NK cells (effectors) andmouse or human cancer cells (target cells). Mouse NK cells are isolatedfrom peripheral blood, bone marrow, or spleens; human NK cells areisolated from peripheral blood. Human cancer cell lines or primarysamples are labeled for use as target cells (e.g. with chromium orfluorescent dye).

The NK cells and cancer cells are combined in vitro, and co-culture withthe following treatments:

-   -   Vehicle control (e.g. PBS)    -   CD137 agonist alone    -   CD47 antagonist alone    -   CD47 antagonist plus CD137 agonist    -   Tumor-binding antibody alone    -   Tumor-binding antibody+CD137 agonist alone    -   Tumor-binding antibody+CD47 antagonist alone    -   Tumor-binding antibody+CD47 antagonist plus CD137 agonist

ADCC is measured via chromium-release assay or flow cytometry cell deathassays (e.q. Annexin V/DAPI staining). NK cell cytokine (e.g. IFN-gamma)release is measured via ELISA. The change in cell death and cytokinerelease in the presence of tumor-binding antibody+CD47 antagonist plusCD137 agonist is determined relative to the mono-therapies and dualtherapies listed above.

Example 3 In Vivo Experiment Protocol

Cancer cells are injected into mice via subcutaneous, retroperitoneal,or peripheral blood injection and allowed to engraft. The animals arerandomized into four treatment groups:

1. Vehicle control (e.g. PBS)

2. CD137 agonist alone

3. CD47 antagonist alone

4. CD47 antagonist plus CD137 agonist

Mice are treated daily, three times per week, twice per week, or onceper week with the respective treatments. Tumor burden is measured bytumor volume measurements, bioluminescence using labeled cancer cells(e.g. luciferase positive cells), and/or analysis of peripheral blood.CD137 expression on NK cells is measured in peripheral blood and intumors. The overall survival of the mice is also measured.

Example 4 In Vivo Experiment Protocol for Synergy with Tumor-SpecificMonoclonal Antibodies

Cancer cells are injected into mice via subcutaneous, retroperitoneal,or peripheral blood injection and allowed to engraft. The animals arerandomized into four treatment groups:

1. Tumor-binding antibody alone

2. Tumor-binding antibody+CD137 agonist alone

3. Tumor-binding antibody+CD47 antagonist alone

4. Tumor-binding antibody+CD47 antagonist plus CD137 agonist

Mice are treated daily, three times per week, twice per week, or onceper week with the respective treatments. Tumor burden is measured bytumor volume measurements, bioluminescence using labeled cancer cells(e.g. luciferase positive cells), and/or analysis of peripheral blood.CD137 expression on NK cells is measured in peripheral blood and intumors. The overall survival of the mice is also measured.

Specific combinations of antibodies and tumors include withoutlimitation the following:

-   -   Engraft mouse mastocytoma P815 cells into DBA/2 mice, treat with        ACK2 (anti-mouse c-kit antibody) CD47/CD137 targeting therapies.    -   Engraft TUBO-EGFR cells into wild-type mice, treat with        cetuximab (anti-human EGFR) and CD47/CD137 targeting therapies.    -   Engraft human melanoma SK-Mel-3 cells into RAG−/− mice, treat        with SR-1 (anti-human c-kit) and CD47/CD137 targeting therapies.    -   Engraft B16 melanoma cells into wild-type mice, treat with TA99        (antibody targeting mouse B16 cells) and CD47/CD137 targeting        therapies.    -   Engraft A20 mouse lymphoma cells into wild-type mice, treat with        anti-mouse CD20 antibody and CD47/CD137 targeting therapies.

Each publication cited in this specification is hereby incorporated byreference in its entirety for all purposes.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appended claims

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

What is claimed is:
 1. A method of targeting cells for immunodepletion,the method comprising: contacting a population of cells comprising thetargeted cells and a population of immune cells with (i) an agent thatblockades CD47 activity; and (ii) an agent that agonizes an immunecostimulatory molecule; in a dose effective to increase depletion of thetargeted cells.
 2. The method of claim 1, wherein the cells are tumorcells.
 3. The method of claim 1 or claim 2, wherein the immune cellscomprise one or both of NK cells and phagocytic cells.
 4. The method ofclaim 3, wherein the immune cells comprise NK cells and macrophages. 5.The method of any one of claims 1-4, wherein the contacting is performedon a mammal in vivo.
 6. The method of any one of claims 1-5, whereindepletion of the target cells is enhanced relative to the depletionobserved with a monotherapy of (i) an agent that blockades CD47activity; or (ii) an agent that agonizes an immune costimulatorymolecule.
 7. The method of any one of claims 1-6, wherein the immunecostimulatory molecule is CD137.
 8. The method of any one of claims 1-6,wherein the immune costimulatory molecule is OX40.
 9. The method of anyone of claims 1-6, wherein the immune costimulatory molecule is CD30.10. The method of any one of claims 1-6, wherein the immunecostimulatory molecule is glucocorticoid-induced TNFR-related (GITR)protein.
 11. The method of any one of claims 1-6, wherein the immunecostimulatory molecule is immune costimulatory protein (ICOS).
 12. Themethod of any one of claims 1-11, wherein the agent that agonizes animmune costimulatory molecule is an antibody.
 13. The method of any ofclaims 1-11, wherein the agent that agonizes an immune costimulatorymolecule is a ligand of the molecule.
 14. The method of any one ofclaims 1-13, further comprising contacting the cell population with(iii) an agent that specifically binds to the targeted cells.
 15. Themethod of claim 14, wherein the agent that specifically binds to thetargeted cells is a tumor-specific antibody.
 16. The method of any ofclaims 1-15 wherein the agent that blockades CD47 activity comprises asoluble SIRPα polypeptide.
 17. The method of claim 16, wherein thesoluble SIRPα polypeptide is a high affinity SIRPα variant.
 18. Themethod of claim 17, wherein the soluble SIRPα polypeptide is CV1. 19.The method of any one of claims 16-18, wherein the agent is a fusionprotein comprising a SIRPα polypeptide.
 20. The method of claim 19,wherein the agent is a monomer or a dimer.
 21. The method of any ofclaims 1-15 wherein the agent that agent that blockades CD47 activity isan anti-CD47 antibody.
 22. The method of any of claims 1-15 wherein theagent that agent that blockades CD47 activity is an anti-SIRPα antibody23. The method of claim 21, wherein the anti-CD47 antibody comprises anIgG4 Fc region.
 24. The method of claim 21 wherein the antibody is5F9-G4.
 25. The method of any of claims 1-24, wherein said mammal is amouse.
 26. The method of any of claims 1-24, wherein said mammal is ahuman.